Project Summary The ability of the cell to control progression through the cell cycle in a regulated manner is of fundamental importance to eukaryotic biology. Breakdown of the checkpoints controlling the cell cycle results in catastrophic effects on normal cell function and is a defining feature of cancer cells. Cell division cycle 34 (Cdc34) is a ubiquitin (Ub) conjugating enzyme (E2) that plays an essential role in controlling progression of the cell cycle at both the G1/S and G2/M checkpoints by ubiquitinating key cell cycle regulatory proteins and signaling for their proteasomal destruction at defined timepoints. E2 enzymes such as Cdc34 are the central players in the Ub conjugation cascade in which the sequential interactions and activities of three enzymes (E1, E2, and E3) are required for ubiquitination of target proteins. This cascade is initiated by E1, which activates and transfers Ub to tens of different E2s. The resulting E2~Ub intermediates next interact with a repertoire of hundreds of Ub E3 ligases that catalyze Ub conjugation to target proteins by distinct mechanisms. While in most cases the cognate E2/E3 pair responsible for regulation of a particular biological process is unknown, the Skp, Cullin, F-box (SCF) subfamily of RING E3 ligases is well- established to function with Cdc34 to control cell cycle progression by catalyzing the extension of Lysine 48- linked polyubiquitin (polyUb) chains on target proteins that serve as a signal for proteasomal destruction. Despite its fundamental importance, many key questions remain about how Cdc34 performs its functions in the cell including: 1) how Cdc34 is recruited to E1 during formation of the Cdc34~Ub intermediate, 2) how Cdc34~Ub is subsequently recruited to SCF, and 3) how the Cdc34/SCF complex specifically catalyzes Lys48-linked polyUb chains that signal for proteasomal destruction of target proteins as opposed to other types of polyUb chains. Cdc34 harbors a number of unique sequence and structural features compared to other E2s that are crucial for its function, though the structural basis for these observations are unknown. These features include an acidic loop insertion proximal to the active site and a large C-terminal extension, both of which are required for Lys48-linked polyUb chain specificity and maximal enzymatic activity. Through use of structural, biochemical/biophysical, and cell-based approaches, this proposal aims to establish the rules governing molecular recognition in E1/Cdc34 and Cdc34/SCF interactions (Aim 1) and to determine the structural basis by which Cdc34 and SCF function together to specifically assemble Lys48- linked polyUb chains (Aim 2). Ub signaling is a validated target for therapeutic intervention in cancer with FDA-approved medications targeting the proteasome currently extending the lives of multiple myeloma patients. A molecular understanding of how E1, Cdc34, and SCF work together to control the cell cycle resulting from the proposed studies could provide a platform for the development of novel cancer therapeutics.